Interpretive Summary: Plants defend themselves from microbial and insect pests by producing a number of different compounds that are toxic to the invading organism. However, these defense mechanisms are often inadequate to protect the plant and need to be supplemented with synthetic, chemical pesticides. We have genetically transformed cotton plants by introducing a gene that enables them to produce a small protein or peptide that has been shown in our laboratory to be toxic to fungal and bacterial plant pathogens. The peptide does not harm mammalian cells at the levels required to kill microbial pathogens. We identified cotton plants carrying the peptide gene and many of these plants demonstrated increased resistance to attack by both bacterial and fungal pathogens. In addition to reducing toxins produced by fungi and bacteria, successful expression of the peptide gene in commercially important varieties of cotton and other crops, such as corn, peanuts, and tree nuts, will reduce the need for treating these crops with chemical pesticides thus reducing production costs for farmers. Less dependence on chemical pesticides will also be of benefit to the environment.

Technical Abstract:
Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium-mediated transformation. PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts. In vitro assays with crude leaf protein extracts from R0 and R1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI121. Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats. In planta assays with the fungal pathogen, Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in negative control R1 seedlings (pBI121-transformed), while transgenic R1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen. Significant advantages of synthetic peptides in developing transgenic crop plants that are resistant to diseases and mycotoxin-causing fungal pathogens are highlighted in this report.